A. Field of the Invention
The present invention relates generally to the field of molecular biology, and particularly to the area of natural and synthetic peptides. More particularly, the invention discloses HER-2/neu peptide, DNA segment, antibody compositions. Various methods for making and using these compositions are disclosed, such as, for example, the use of peptides and antibodies in various pharmacological and immunological applications, including the stimulation of cytotoxic T-lymphocytes and cancer therapies.
B. Description of the Related Art
1. HER-2/neu Proto-Oncogene
The HER-2/neu proto-oncogene (HER-2) encodes a transmembrane protein whose expression is enhanced in a number of breast and ovarian tumors and correlates with tumor aggressiveness. Because of its expression on normal epithelial cells, HER-2 can be defined as a tumor-associated antigen (Ag) and may be of interest as a target of a therapeutic anti-tumor T-cell response. A CD3+CD8+CD4xe2x88x92 line isolated from cell cultures has been shown to lyse HLA-A2+, HER-s+ ovarian tumors but not natural killer (NK) target K562 cells, and showed significantly higher lysis of HER-2high than of HER-2low ovarian tumors. Some inhibition of lysis was inhibited by HER-2 peptide-pulsed HLA-A2+ targets, suggesting that some epitopes may be present on tumor cells associated with HLA-A2.
2. Tumor-Reactive T-Cells
Tumor reactive T-cells have been reported to mediate therapeutic responses against human cancers (Rosenberg et al., 1988). In certain instances, in human immunotherapy trials with tumor infiltrating lymphocytes (TIL) or tumor vaccines, these responses correlated either with in vitro cytotoxicity levels against autologous tumors (Aebersold et al., 1991) or with expression of certain HLA-A,B,C gene products (Marincola et al., 1992). Recent studies (Ioannides et al., 1992) have proposed that in addition to virally encoded and mutated oncogenes, overexpressed self-proteins may elicit some degree of tumor-reactive cytotoxic T-lymphocytes (CTLs) in patients with various malignancies (Ioannides et al., 1992; Ioannides et al., 1993; Brichard et al., 1993; Jerome et al., 1991). Autologous tumor reactive CTLs can be generated from lymphocytes infiltrating ovarian malignant ascites (Ioannides et al., 1991), and overexpressed proteins such as HER-2 may be targets for CTL recognition (Ioannides et al., 1992).
Information on epitopes of self-proteins recognized in the context of MHC Class I molecules remain limited, despite a few attempts to identify epitopes capable of in vitro priming and Ag-specific expansion of human CTLs. For example, peptide epitopes have been proposed which are likely candidates for binding on particular MHC Class I Ag (Falk et al., 1991), and some studies have attempted to define peptide epitopes which bind MHC Class I antigens.
Short synthetic peptides have been used either as target antigens for epitope mapping or for induction of in vitro primary and secondary CTL responses to viral and parasitic Ags (Bednarek et al., 1991; Gammon et al., 1992; Schmidt et al., 1992; Kos and Mxc3xcllbacher, 1992; Hill et al., 1992). Unfortunately, these studies failed to show the ability of proto-oncogene peptide analogs to stimulate in vitro human CTLs to lyse tumors endogenously expressing these antigens.
3. Synthetic Peptides and T-Cell Epitope Mapping
Synthetic peptides have been shown to be a useful tool for T-cell epitope mapping. However in vivo and in vitro priming of specific CTLs has encountered difficulties (Alexander et al., 1991; Schild et al., 1991; Carbone et al., 1988). It is generally considered that in vitro CTL priming cannot necessarily be achieved with peptide alone, and in fact, a high antigen density is thought to be required for peptide priming (Alexander et al., 1991). Even in the limited instances when specific priming was achieved, APC or stimulators were also required at high densities (Alexander et al., 1991).
It is not clear when CTL induction by HER-2 peptides in vitro was observed whether this reflects secondary activation of CTL specific for, or cross-reacting with, the Ag of interest. Whether or not this cross-reactivity can constitute the foundation for development of an in vitro CTL response to tumor remains to be determined.
Therefore, what is lacking in the prior art are universal epitopes which are both immunodominant and CTL-stimulating. Moreover, methods for the use of such CTL-stimulating peptides would be most desirable in the treatment of human cancers, particularly of breast and ovarian etiology, and the development of cancer vaccines. Identifying universal oncoprotein epitopes would permit not only an increased understanding of tumor immunity and autoimmunity in humans, but would also open the door to the design of novel therapeutic strategies for proliferative cell disorders such as human cancers, and particularly breast and ovarian cancers.
The present invention seeks to overcome these and other inherent deficiencies in the prior art by providing the identification of native and synthetic proteins or peptides derived from the HER-2/neu proto-oncogene gene product, and methods for their use in stimulating cytotoxic T-lymphocytes. These selected xe2x80x9cuniversalxe2x80x9d immunodominant epitopic peptides, and their synthetically-optimized derivatives are envisioned to be useful in the development of tumor vaccines, and anti-cancer therapeutics. Pharmaceutical reagents resulting from these novel peptides and the DNA segments which encode them will also likely prove useful as test reagents for the detection of HER-2/neu-related polypeptides, facilitate the production of anti-peptide antibodies specific to a range of HER-2/neu-related polypeptides, and result in the stimulation and production of cytotoxic T-lymphocytes specific for a variety of proliferative disorders including human cancer.
Synthetic peptide analogs can be used to define CTL epitopes recognized by tumor reactive T-cells and to stimulate in vitro peptide-specific CTLs. Such CTLs can be further evaluated for recognition of targets endogenously expressing the particular antigen (Ag) and for Ag-specific adoptive therapy.
Disclosed herein are compositions and methods for their making and use in development of anti-cancer vaccines. The generation in vitro of HLA-A2-restricted CTLs using HER-2 synthetic peptide analogs as immunogens, and peripheral blood mononuclear cells (PBMC) from healthy volunteers as responder cells is also described. Lysis with isolated CD8+ T-cells from these CTL cultures was observed using both HER-2 peptide-pulsed HLA-A2 from these CTL cultures was observed using both HER-2 peptide-pulsed HLA-A2 transfectants and HLA-A2+ ovarian tumors expressing high levels of HER-2 as targets.
Another aspect of the invention is the development and maintenance in long-term culture a CD3+CD8+CD4xe2x88x92 line by restimulation with HER-2 peptide-pulsed autologous PBMC. This line lysed HLA-A2+, HER-2high ovarian tumors, but not HLA-A2+, HER-2low ovarian tumors. Tumor lysis was inhibited by HER-2 peptide-pulsed HLA-A2+ transfectants, demonstrating that epitopes either similar or cross-reactive with the ones recognized by CTLs on the peptide used as immunogen in vitro are present on the tumor cells. These CTL showed lower lysis of targets pulsed with unrelated peptides (analogs of Muc-1 core peptide where HLA-A2 anchors were introduced).
A novel approach to developing tumor reactive CTLs is disclosed which focuses on a target Ag expressed on the tumor of interest and identifying CTLs induced in vivo or developed in vitro that recognize this target Ag. In tumor cells the level of expression of a particular protein may be 102-103 fold higher than in normal tissue.
The inventors expect that a number of target T-cell Ags on human tumors may be derived from proteins that are expressed at low levels in normal cells, and at significantly higher concentration in tumor cells, such as overexpressed proto-oncogene products (Ioannides et al., 1992). The rationale for this hypothesis is: first, peptides from self-proteins which fulfill the criteria of MHC allele-specific motifs should be capable of binding to the Ag binding pockets in the MHC class I heavy chain; and second, positive and negative selection of T-cell repertoire may result in elimination or tolerization of high-affinity self-reactive CTLs (Parmianai, 1993), although such peptide-MHC complexes should have lower affinity for the TCR than a de novo expressed epitope from a self-protein (as a consequence either of mutations creating HLA-anchors or modifying the core recognized by the TCR), their presence in high concentration may engage a large number of TCR.
The HER-2/neu proto-oncogene was identified because it is overexpressed (in certain instances by several hundred fold) in a number of breast and ovarian tumors (Slamon et al., 1989). Moreover, it was found that several CTL-TAL lines isolated from ovarian malignant ascites could lyse autologous ovarian tumors.
Surprisingly, the inventors also discovered that this lysis could also be effectively inhibited by natural and synthetic peptide analogs of HER-2. These results suggested that these novel peptides acted as epitopes that were either derived from an endogenously-processed HER-2 peptide, mimicked, or cross-reacted with a peptide of related sequence derived from another protein.
Novel synthetic peptide compositions have also been developed which correspond to the HER-2:968-981 and 971-979 regions. The compositions disclosed herein, were found to stimulate in vitro PBMCs from healthy HLA-A2+ human volunteers (Fisk et al., 1994), and CTLs (induced by peptide stimulation) consequently lysed tumors overexpressing HER-2 (Fisk et al., 1994). These studies demonstrated that these CTLs can effectively recognize the epitope peptides of the present invention, and that these HER-2-derived peptides can stimulate in vitro PBMCs to induce peptide reactive CTLs.
This possibility may be particularly relevant for induction of Ag and tumor-specific CTLs because peripheral T-cells that can recognize such peptides from non-mutated self proteins are those that have either escaped elimination or may have become tolerant to one or more of these antigenic epitopes due to low affinity TCR-MHC interactions (Ioannides et al., 1992; Parmiani, 1993).
Other aspects of this invention include the identification of candidate HER-2-derived T-cell epitopes based on the presence of anchors for HLA-A2, the analysis of these peptides to affect the conformation of HLA-A2 as an indication of peptide binding, and finally, the demonstration that these peptides can stimulate in vitro peptide reactive CTLs from human HLA-A2+ PBMC.
Methods are described herein for stimulation of CTLs (and consequently, production of an immune response) employing the novel compositions disclosed herein. In vitro induction of cellular responses to the peptides of the present invention by PBMC from healthy HLA-A2+ volunteers demonstrated their ability to stimulate and/or restimulate pre-existing T-cell responses to HER-2. The peptides induced proliferative responses in one of four donors tested and CTL responses (one of three peptides tested in two of three donors), and may be used to induce tumor-reactive T-cells in vitro and in vivo through either peptide-, lipopeptide-, or cell-mediated methods. These peptides therefore find utility in both generating an immune response, and serving as antigens in the preparation of peptide-specific antibodies.
The peptides of this invention also may be used in embodiments involving treatment, diagnosis, and identification of proliferative cell disorders such as cancer, and particularly cancers such as, inter alia, breast and ovarian tumors. Methods of identification of HER-2/neu-containing cells, and also neu-related proto-oncogene and oncogene products are also disclosed.
Cancer treatment methods, including vaccine development are another aspect of the present invention. Additionally, a variety of in vitro and in vivo assay protocols are facilitated as a result of the novel compositions disclosed herein. In addition to stimulating CTLs, and generating an immune response in an animal, and particularly in a human, the peptides may also be used as immunogens to generate anti-peptide antibodies, which themselves have many uses, not least of which is the detection of oncogene-containing cells (e.g., detection of HER-2/neu, related oncogenic polypeptides, or peptide fragments thereof, in diagnostic tests and kits based upon immunological binding assays).
Also, since the peptides of the invention bind to T-cells, they may be employed in assays to identify T-cells, and particularly CTLs, for example, to assess the immunological capacity of a given individual or animal, or even to purify CTLs themselves. Such methods could utilize radioactively- or enzymatically-labeled peptides or anti-peptide antibodies, such as those described herein.
Therefore, one contemplated use for the described peptides concerns their use in methods for detecting the presence of T-cells within a sample. These methods include contacting a sample suspected of containing T-cells with a peptide or composition in accordance with the present invention under conditions effective to allow the peptide(s) to form a complex with T-cells of the sample. One then detects the presence of the complex by detecting the presence of the peptide(s) within the complex, e.g., by either originally using radiolabeled peptides or by subsequently employing anti-peptide antibodies and standard secondary antibody detection techniques.
Preferred peptides of the present invention will likely be from about 6 to about 20 amino acids, in length, with peptides of from 7 to about 15 amino acids in length being even more preferred. Most preferred are peptides having lengths of from about 8 to about 10 amino acids in length, with nonameric and decameric peptides being most preferred. These peptides may include one or more D-amino acids, or may even be entirely composed of D-amino acids, and may, of course, contain additional elements, as desired for stability or even for targeting purposes.
The peptides, or multimers thereof, may be dispersed in any one of the many pharmacologically-acceptable vehicles known in the art and particularly exemplified herein. As such, the peptides may be encapsulated within liposomes or incorporated in a biocompatible coating designed for slow-release. The preparation and use of appropriate therapeutic formulations will be known to those of skill in the art in light of the present disclosure. The peptides may also be used as part of a prophylactic regimen designed to prevent, or protect against, possible cancer progression and/or metastasis and may thus be formulated as a vaccine, particularly as a method of stimulating anti-tumor CTLs.
The present invention also provides methods for identifying HER-2/neu and related proto-oncogene products, which methods comprise contacting the cells suspected of containing such polypeptides with an immunologically effective amount of a composition comprising one or more specific anti-peptide antibodies disclosed herein. Peptides that include the amino acid sequence of any of SEQ ID NO:1 through SEQ ID NO:29 and their derivatives will be preferred for use in generating such anti-CTL-stimulating peptide antibodies.
The invention thus also provides compositions, including peptides, peptide multimers, and pharmaceutical compositions derived therefrom, that contain one or more peptides of from 8 to about 20 amino acids in length that include within their sequence the peptide sequence identified by the formula: AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8; where AA1 is Leu, Met, Ile, or Val; AA2 is any amino acid; AA3 is any amino acid; AA4 is Ser, Glu, Thr, or Tyr; AA5 is any amino acid; AA6 is any amino acid; AA7 is any amino acid; and AA7 is Val, Leu, Met, Ile, or Cys. These peptides are submitted to be capable of stimulating CTLs and producing an immune response in vitro and in vivo.
Another aspect of the present invention concerns the use of the amino acid sequences disclosed herein in the determination of molecular weights of low-molecular-weight polypeptides. These peptides represent a significant improvement over commercially-available protein standards in this area owing to their small size, and the presence of known nonapeptide motifs. Commercially-available standards typically have a range of 3,000 to 200,000 Da, and as such, are not useful in the characterization of proteins having molecular weights of about 300 to about 3,000 Da using either conventional or gradient SDS-PAGE.
In a similar fashion, the peptides, and more particularly peptide oligomers, of the present invention are readily employed as standards in the identification of small molecular-weight polypeptides using chromatographic separation. In preferred embodiments, paper chromatography is utilized and proteins are subsequently visualized after reaction with ninhydrin. More preferred is the use of thin-layer chromatography in either one or two dimensions.
The use of the peptides and peptide motifs of the present invention is also contemplated for the calibration and standardization of chromatographic columns used in the separation of low-molecular-weight polypeptides. These peptides, and multimers thereof, find important use in the calibration of low-molecular-weight-range columns. Such molecular sieve (or gel filtration) chromatography columns may include a filtration medium having the capacity to fractionate any protein of interest and the peptides of the present invention. Preferred chromatographic media would include any gel filtration medium having a molecular fractionation range suitable for the particular protein of interest. Preferred media would include the G-50 or G-25 Sephadex(copyright) resins which have an approximate fractionation range of 1,500-30,000 and 100-5,000 Da, respectively. A more preferred medium would be either the G-10 or G-15 Sephadex(copyright) resins which have an approximate fractionation range of 0-700 and 0-1500 Da, respectively.
Peptides of the present invention comprising aromatic amino acids and multimers thereof may also be used as protein concentration standards in reactions employing either the Folin reagent (Lowry et al., 1951), the biuret reaction (Coakley and James, 1978) or the bicinconinic acid assay (Pierce Chemical Corp., Rockford, Ill.). Peptides and multimers thereof lacking aromatic amino acids may also be used as protein concentration standards in the latter two reactions.